Lipids Modulate the Increase of BK Channel Calcium Sensitivity by the β1 Subunit

Co-expression of the auxiliary β1 subunit with the pore forming α subunit of BK dramatically alters apparent calcium sensitivity. Investigation of the mechanism underlying the increase in calcium sensitivity of BK in smooth muscle has concentrated on the energetic effect of β1′s interaction with α. We take a novel approach, exploring whether β1 modification of calcium sensitivity reflects altered interaction between the channel protein and surrounding lipids. We reconstituted hSlo BK α and BK α+β1 channels into two sets of bilayers. One set contained POPE with POPS, POPG, POPA and POPC, where the length of acyl chains is constant, but surface charge differs. The second set is a series of neutral bilayers formed from DOPE with phosphatidylcholines (PCs) of varying acyl chain lengths: C (14∶1), C (18∶1), C (22∶1) and C (24∶1), and with brain sphingomyelin (SPM), in which surface charge is constant, but bilayer thickness varies. The increase in calcium sensitivity caused by the β1 subunit was preserved in negatively charged lipid bilayers but not in neutral bilayers, indicating that modification of apparent Ca²⁺ sensitivity by β1 is modulated by membrane lipids, requiring negatively charged lipids in the membrane. Moreover, the presence of β1 reduces BK activity in thin bilayers of PC 14∶1 and thick bilayers containing SPM, but has no significant effect on activity of BK in PC 18∶1, PC 22∶1 and PC 24∶1 bilayers. These data suggest that auxiliary β1 subunits fine-tune channel gating not only through direct subunit-subunit interactions but also by modulating lipid-protein interactions.     

11,12-EET Stimulates the Association of BK Channel α and β1 Subunits in Mitochondria to Induce Pulmonary Vasoconstriction

In the systemic circulation, 11,12-epoxyeicosatrienoic acid (11,12-EET) elicits nitric oxide (NO)- and prostacyclin-independent vascular relaxation, partially through the activation of large conductance Ca²⁺-activated potassium (BK) channels. However, in the lung 11,12-EET contributes to hypoxia-induced pulmonary vasoconstriction. Since pulmonary artery smooth muscle cells also express BK channels, we assessed the consequences of BKβ₁ subunit deletion on pulmonary responsiveness to 11,12-EET as well as to acute hypoxia. In buffer-perfused mouse lungs, hypoxia increased pulmonary artery pressure and this was significantly enhanced in the presence of NO synthase (NOS) and cyclooxygenase (COX) inhibitors. Under these conditions the elevation of tissue EET levels using an inhibitor of the soluble epoxide hydrolase (sEH-I), further increased the hypoxic contraction. Direct administration of 11,12-EET also increased pulmonary artery pressure, and both the sEH-I and 11,12-EET effects were prevented by iberiotoxin and absent in BKβ₁ ^−/− mice. In pulmonary artery smooth muscle cells treated with NOS and COX inhibitors and loaded with the potentiometric dye, di-8-ANEPPS, 11,12-EET induced depolarization while the BK channel opener NS1619 elicited hyperpolarization indicating there was no effect of the EET on classical plasma membrane BK channels. In pulmonary artery smooth muscle cells a subpopulation of BK channels is localized in mitochondria. In these cells, 11,12-EET elicited an iberiotoxin-sensitive loss of mitochondrial membrane potential (JC-1 fluorescence) leading to plasma membrane depolarization, an effect not observed in BKβ₁ ^−/− cells. Mechanistically, stimulation with 11,12-EET time-dependently induced the association of the BK α and β₁ subunits. Our data indicate that in the absence of NO and prostacyclin 11,12-EET contributes to pulmonary vasoconstriction by stimulating the association of the α and β₁ subunits of mitochondrial BK channels. The 11,12-EET-induced activation of BK channels results in loss of the mitochondrial membrane potential and depolarization of the pulmonary artery smooth muscle cells.     

Is the Volume-Regulated Anion Channel VRAC a “Water-Permeable” Channel?

The volume-regulated anion channel, VRAC, plays an important role in cell volume regulation. This channel is permeable for a wide variety of anions, amino acids, and organic osmolytes, including taurine. However, nothing is known about possible water permeability of this channel. Water permeability of endothelial cells is estimated from the initial rate of cell swelling because of a hypotonic challenge. As a result of simultaneous volume and current measurements, it will be shown that water permeability is decreased by inhibition of VRAC. It is concluded that water permeates VRAC and might be able to accelerate water transport by providing an additional permeation pathway for water. Therefore VRAC can be considered as a water-permeable, "wet" channel.     

Positions of β2 and β3 subunits in the large-conductance calcium- and voltage-activated BK potassium channel

Large-conductance voltage- and Ca²⁺-gated K⁺ channels are negative-feedback regulators of excitability in many cell types. They are complexes of α subunits and of one of four types of modulatory β subunits. These have intracellular N- and C-terminal tails and two transmembrane (TM) helices, TM1 and TM2, connected by an ∼100-residue extracellular loop. Based on endogenous disulfide formation between engineered cysteines (Cys), we found that in β2 and β3, as in β1 and β4, TM1 is closest to αS1 and αS2 and TM2 is closest to αS0. Mouse β3 (mβ3) has seven Cys in its loop, one of which is free, and this Cys readily forms disulfides with Cys substituted in the extracellular flanks of each of αS0–αS6. We identified by elimination mβ3-loop Cys152 as the only free Cys. We inferred the disulfide-bonding pattern of the other six Cys. Using directed proteolysis and fragment sizing, we determined this pattern first among the four loop Cys in β1. These are conserved in β2–β4, which have four additional Cys (eight in total), except that mβ3 has one fewer. In β1, disulfides form between Cys at aligned positions 1 and 8 and between Cys at aligned positions 5 and 6. In mβ3, the free Cys is at position 7; position 2 lacks a Cys present in all other β2–β4; and the disulfide pattern is 1–8, 3–4, and 5–6. Presumably, Cys 2 cross-links to Cys 7 in all other β2–β4. Cross-linking of mβ3 Cys152 to Cys substituted in the flanks of αS0–S5 attenuated the protection against iberiotoxin (IbTX); cross-linking of Cys152 to K296C in the αS6 flank and close to the pore enhanced protection against IbTX. In no case was N-type inactivation by the N-terminal tail of mβ3 perturbed. Although the mβ3 loop can move, its position with Cys152 near αK296, in which it blocks IbTX binding, is likely favored.     

Aminopyridines Potentiate Synaptic and Neuromuscular Transmission by Targeting the Voltage-activated Calcium Channel �� Subunit

Aminopyridines such as 4-aminopyridine (4-AP) are widely used as voltage-activated K⁺ (Kv) channel blockers and can improve neuromuscular function in patients with spinal cord injury, myasthenia gravis, or multiple sclerosis. Here, we present novel evidence that 4-AP and several of its analogs directly stimulate high voltage-activated Ca²⁺ channels (HVACCs) in acutely dissociated neurons. 4-AP, 4-(aminomethyl)pyridine, 4-(methylamino)pyridine, and 4-di(methylamino)pyridine profoundly increased HVACC, but not T-type, currents in dissociated neurons from the rat dorsal root ganglion, superior cervical ganglion, and hippocampus. The widely used Kv channel blockers, including tetraethylammonium, α-dendrotoxin, phrixotoxin-2, and BDS-I, did not mimic or alter the effect of 4-AP on HVACCs. In HEK293 cells expressing various combinations of N-type (Cav2.2) channel subunits, 4-AP potentiated Ca²⁺ currents primarily through the intracellular β₃ subunit. In contrast, 4-AP had no effect on Cav3.2 channels expressed in HEK293 cells. Furthermore, blocking Kv channels did not mimic or change the potentiating effects of 4-AP on neurotransmitter release from sensory and motor nerve terminals. Thus, our findings challenge the conventional view that 4-AP facilitates synaptic and neuromuscular transmission by blocking Kv channels. Aminopyridines can directly target presynaptic HVACCs to potentiate neurotransmitter release independent of Kv channels.     

Differential efficacy of GoSlo-SR compounds on BKα and BKαγ1–4 channels

Large conductance, voltage and Ca²⁺ activated K⁺ channels (BK channels) are abundantly expressed throughout the body and are important regulators of smooth muscle tone and neuronal excitability. Their dysfunction is implicated in various diseases including overactive bladder, hypertension and erectile dysfunction. Therefore, BK channel openers bear significant therapeutic potential to treat the above diseases. GoSlo-SR compounds were designed to be potent and efficacious BK channel openers. Although their structural activity relationships, activation in both BKα and BKαβ channels and the hypothetical mode of action of these compounds has been studied in detail in recent years, their effectiveness to open the BKαγ channels still remains unexplored. In this study, we have examined the efficacy of 3 closely related GoSlo-SR openers, GoSlo-SR-5-6 (SR-5-6), GoSlo-SR-5-44 (SR-5-44) and GoSlo-SR-5-130 (SR-5-130) using inside out patches on BKα channels coexpressed with 4 different LRRC (γ_1–4) subunits in HEK293 cells. Our data suggests that the activation effects due to SR-5-6 were not significantly affected in the presence of γ_1–4 subunits. Interestingly, the effects of more efficacious BK channel opener SR-5-44 were altered by different γ subunits. In cells expressing BKα channels, the shift in V_1/2 (ΔV_1/2) induced by SR-5-44 (3 μM) was ?76 ± 3 mV, whereas it was significantly reduced by ～70 % in BKαγ₁ channels (ΔV_1/2= ?23 ± 3, p < 0.001, ANOVA). In BKαγ₂ channels the ΔV_1/2 was ?36 ± 1 mV, which was less than that observed in BKαγ₃ and BKαγ₄ channels where the ΔV_1/2 was ?47 ± 5 mV, and ?82 ± 5 mV, respectively. Additionally, the excitatory effects of a ‘β specific’ BK channel opener, SR-5-130 were only partially restored in the patches containing BKαγ_1–4 channels. Together this data highlights that subtle modifications in GoSlo-SR structures alter their effectiveness on BK channels with accessory γ subunits and this study might provide a scaffold for the development of more tissue specific BK channel openers.     

Acetylcholine-activated Cl? Channel in the Chara Tonoplast

Acetylcholine has long been suggested to play a role in controlling physiological processes in plants, but no mechanism has been shown for its action. We show here that a chloride channel in the tonoplast (vacuolar membrane) of Chara corallina responds to acetylcholine. The channel has a conductance of 45 pS. The effect of acetylcholine is enhanced by nicotine, with the open probability increasing from 0.05 in the presence of 4 mM acetylcholine to 0.3 in the presence of 4 mM acetylcholine + 6 mM nicotine. Some effects of acetylcholine were seen at concentrations as low as 20 microM, with a maximum effect between 1 and 10 mM. In the intact cell, acetylcholine prolongs the depolarized phase of the action potential. We propose that this acetylcholine-gated channel has evolved separately from the mammalian acetylcholine-gated channel, and suggest that this represents a third form of acetylcholine signal transduction, after the nicotinic and muscarinic pathways in animal systems.     

Structures and Functions of Calcium Channel β Subunits

Calcium channel subunits have profound effects on how ₁ subunits perform. In this article we summarize our present knowledge of the primary structures of subunits as deduced from cDNAs and illustrate their different properties. Upon co-expression with ₁ subunits, the effects of subunits vary somewhat between L-type and non-L-type channels mostly because the two types of channels have different responses to voltage which are affected by subunits, such as long-lasting prepulse facilitation of _1C (absent in _1E) and inhibition by G protein dimer of _1E, absent in _1C. One subunit, a brain 2a splice variant that is palmitoylated, has several effects not seen with any of the others, and these are due to palmitoylation. We also illustrate the finding that functional expression of ₁ in oocytes requires a subunit even if the final channel shows no evidence for its presence. We propose two structural models for Ca²⁺ channels to account for ₁ alone channels seen in cells with limited subunit expression. In one model, dissociates from the mature ₁ after proper folding and membrane insertion. Regulated channels seen upon co-expression of high levels of would then have subunit composition ₁. In the other model, the chaperoning remains associated with the mature channel and ₁ alone channels would in fact be ₁ channels. Upon co-expression of high levels of the regulated channels would have composition [₁].     

Orientation of the Calcium Channel β Relative to the α12.2 Subunit Is Critical for Its Regulation of Channel Activity

Background

The Ca_vβ subunits of high voltage-activated Ca²⁺ channels control the trafficking and biophysical properties of the α₁ subunit. The Ca_vβ-α₁ interaction site has been mapped by crystallographic studies. Nevertheless, how this interaction leads to channel regulation has not been determined. One hypothesis is that βs regulate channel gating by modulating movements of IS6. A key requirement for this direct-coupling model is that the linker connecting IS6 to the α-interaction domain (AID) be a rigid structure.

Methodology/Principal Findings

The present study tests this hypothesis by altering the flexibility and orientation of this region in α₁2.2, then testing for Ca_vβ regulation using whole cell patch clamp electrophysiology. Flexibility was induced by replacement of the middle six amino acids of the IS6-AID linker with glycine (PG6). This mutation abolished β2a and β3 subunits ability to shift the voltage dependence of activation and inactivation, and the ability of β2a to produce non-inactivating currents. Orientation of Ca_vβ with respect to α₁2.2 was altered by deletion of 1, 2, or 3 amino acids from the IS6-AID linker (Bdel1, Bdel2, Bdel3, respectively). Again, the ability of Ca_vβ subunits to regulate these biophysical properties were totally abolished in the Bdel1 and Bdel3 mutants. Functional regulation by Ca_vβ subunits was rescued in the Bdel2 mutant, indicating that this part of the linker forms β-sheet. The orientation of β with respect to α was confirmed by the bimolecular fluorescence complementation assay.

Conclusions/Significance

These results show that the orientation of the Ca_vβ subunit relative to the α₁2.2 subunit is critical, and suggests additional points of contact between these subunits are required for Ca_vβ to regulate channel activity.     

Large Conductance Voltage- and Ca2+-gated Potassium (BK) Channel β4 Subunit Influences Sensitivity and Tolerance to Alcohol by Altering Its Response to Kinases

Tolerance is a well described component of alcohol abuse and addiction. The large conductance voltage- and Ca²⁺-gated potassium channel (BK) has been very useful for studying molecular tolerance. The influence of association with the β4 subunit can be observed at the level of individual channels, action potentials in brain slices, and finally, drinking behavior in the mouse. Previously, we showed that 50 mm alcohol increases both α and αβ4 BK channel open probability, but only α BK develops acute tolerance to this effect. Currently, we explore the possibility that the influence of the β4 subunit on tolerance may result from a striking effect of β4 on kinase modulation of the BK channel. We examine the influence of the β4 subunit on PKA, CaMKII, and phosphatase modulation of channel activity, and on molecular tolerance to alcohol. We record from human BK channels heterologously expressed in HEK 293 cells composed of its core subunit, α alone (Insertless), or co-expressed with the β4 BK auxiliary subunit, as well as, acutely dissociated nucleus accumbens neurons using the cell-attached patch clamp configuration. Our results indicate that BK channels are strongly modulated by activation of specific kinases (PKA and CaMKII) and phosphatases. The presence of the β4 subunit greatly influences this modulation, allowing a variety of outcomes for BK channel activity in response to acute alcohol.     

Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly

The vertebrate sodium (Na_v) channel is composed of an ion-conducting α subunit and associated β subunits. Here, we report the crystal structure of the human β3 subunit immunoglobulin (Ig) domain, a functionally important component of Na_v channels in neurons and cardiomyocytes. Surprisingly, we found that the β3 subunit Ig domain assembles as a trimer in the crystal asymmetric unit. Analytical ultracentrifugation confirmed the presence of Ig domain monomers, dimers, and trimers in free solution, and atomic force microscopy imaging also detected full-length β3 subunit monomers, dimers, and trimers. Mutation of a cysteine residue critical for maintaining the trimer interface destabilized both dimers and trimers. Using fluorescence photoactivated localization microscopy, we detected full-length β3 subunit trimers on the plasma membrane of transfected HEK293 cells. We further show that β3 subunits can bind to more than one site on the Na_v 1.5 α subunit and induce the formation of α subunit oligomers, including trimers. Our results suggest a new and unexpected role for the β3 subunits in Na_v channel cross-linking and provide new structural insights into some pathological Na_v channel mutations.     

The N-terminal Domain Tethers the Voltage-gated Calcium Channel β2e-subunit to the Plasma Membrane via Electrostatic and Hydrophobic Interactions

The β-subunit associates with the α₁ pore-forming subunit of high voltage-activated calcium channels and modulates several aspects of ion conduction. Four β-subunits are encoded by four different genes with multiple splice variants. Only two members of this family, β_2a and β_2e, associate with the plasma membrane in the absence of the α₁-subunit. Palmitoylation on a di-cysteine motif located at the N terminus of β_2a promotes membrane targeting and correlates with the unique ability of this protein to slow down inactivation. In contrast, the mechanism by which β_2e anchors to the plasma membrane remains elusive. Here, we identified an N-terminal segment in β_2e encompassing a cluster of positively charged residues, which is strictly required for membrane anchoring, and when transferred to the cytoplasmic β_1b isoform it confers membrane localization to the latter. In the presence of negatively charged phospholipid vesicles, this segment binds to acidic liposomes dependently on the ionic strength, and the intrinsic fluorescence emission maxima of its single tryptophan blue shifts considerably. Simultaneous substitution of more than two basic residues impairs membrane targeting. Coexpression of the fast inactivating R-type calcium channels with wild-type β_2e, but not with a β_2e membrane association-deficient mutant, slows down inactivation. We propose that a predicted α-helix within this domain orienting parallel to the membrane tethers the β_2e-subunit to the lipid bilayer via electrostatic interactions. Penetration of the tryptophan side chain into the lipidic core stabilizes the membrane-bound conformation. This constitutes a new mechanism for membrane anchoring among the β-subunit family that also sustains slowed inactivation.     

The Gating Kinetics of the Slow Vacuolar Channel. A Novel Mechanism for SV Channel Functioning?

Although there is consensus that the slow vacuolar or SV channel is a Ca²⁺ release channel, the underlying mechanism of operation is still controversial. The main reason is that the voltage sensitivity of SV gating seems to exclude activation at hyperpolarized (physiological) membrane potentials. Inspired by a study of Gambale et al. (1993) and supported by simulation studies presented here, we interpreted SV activation and deactivation kinetics in terms of a cyclic state diagram originally applied to animal cation-selective channels. A cyclic state diagram allows two pathways of activation operating in opposite directions. One pathway represents the frequently observed slow activation at moderate depolarization (<130 mV). With the open state (O) next to the closed state initially occupied (C ₁), direct transitions from C ₁ to O can account for the fast activation observed at higher depolarized potentials (>130 mV). We hypothesize that similar state transitions directly to O may also occur during hyperpolarization. The implication of this proposed mechanism is that SV accomplishes its physiological role during hyperpolarization-evoked deactivation. Despite their rare occurrence and possibly short duration, these opening events may last long enough to substantially raise the local cytosolic free Ca²⁺ level at the channel mouth by as much as 600 nM/ms. Because under in vivo conditions the Ca²⁺ flux is inwardly directed, the mechanism presented here revives the notion that the SV channel can be subject to calcium-induced calcium release. Present address for H. M.: BioMade Technology Foundation, Nijenborgh 4, 9747 AG Groningen, The Netherlands     

Enhancement effects of martentoxin on glioma BK channel and BK channel (α+β1) subtypes

Background

BK channels are usually activated by membrane depolarization and cytoplasmic Ca²⁺. Especially,the activity of BK channel (α+β4) can be modulated by martentoxin, a 37 residues peptide, with Ca²⁺-dependent manner. gBK channel (glioma BK channel) and BK channel (α+β1) possessed higher Ca²⁺ sensitivity than other known BK channel subtypes.

Methodology and Principal Findings

The present study investigated the modulatory characteristics of martentoxin on these two BK channel subtypes by electrophysiological recordings, cell proliferation and Ca²⁺ imaging. In the presence of cytoplasmic Ca²⁺, martentoxin could enhance the activities of both gBK and BK channel (α+β1) subtypes in dose-dependent manner with EC₅₀ of 46.7 nM and 495 nM respectively, while not shift the steady-state activation of these channels. The enhancement ratio of martentoxin on gBK and BK channel (α+β1) was unrelated to the quantitive change of cytoplasmic Ca²⁺ concentrations though the interaction between martentoxin and BK channel (α+β1) was accelerated under higher cytoplasmic Ca²⁺. The selective BK pore blocker iberiotoxin could fully abolish the enhancement of these two BK subtypes induced by martentoxin, suggesting that the auxiliary β subunit might contribute to the docking for martentoxin. However, in the absence of cytoplasmic Ca²⁺, the activity of gBK channel would be surprisingly inhibited by martentoxin while BK channel (α+β1) couldn''t be affected by the toxin.

Conclusions and Significance

Thus, the results shown here provide the novel evidence that martentoxin could increase the two Ca²⁺-hypersensitive BK channel subtypes activities in a new manner and indicate that β subunit of these BK channels plays a vital role in this enhancement by martentoxin.     

Cys Palmitoylation of the β Subunit Modulates Gating of the Epithelial Sodium Channel

The epithelial Na⁺ channel (ENaC) is comprised of three homologous subunits (α, β, and γ) that have a similar topology with two transmembrane domains, a large extracellular region, and cytoplasmic N and C termini. Although ENaC activity is regulated by a number of factors, palmitoylation of its cytoplasmic Cys residues has not been previously described. Fatty acid-exchange chemistry was used to determine whether channel subunits were Cys-palmitoylated. We observed that only the β and γ subunits were modified by Cys palmitoylation. Analyses of ENaCs with mutant β subunits revealed that Cys-43 and Cys-557 were palmitoylated. Xenopus oocytes expressing ENaC with a β C43A,C557A mutant had significantly reduced amiloride-sensitive whole cell currents, enhanced Na⁺ self-inhibition, and reduced single channel P_o when compared with wild-type ENaC, while membrane trafficking and levels of surface expression were unchanged. Computer modeling of cytoplasmic domains indicated that β Cys-43 is in proximity to the first transmembrane α helix, whereas β Cys-557 is within an amphipathic α-helix contiguous with the second transmembrane domain. We propose that β subunit palmitoylation modulates channel gating by facilitating interactions between cytoplasmic domains and the plasma membrane.     

The HOOK-Domain Between the SH3- and the GK-Domains of CaVβ Subunits Contains Key Determinants Controlling Calcium Channel Inactivation

Ca_Vβ subunits of voltage-gated calcium channels contain two conserved domains, a src-homology-3 (SH3)-domain and a guanylate kinase-like (GK)-domain. The SH3-domain is split, with its final (5th) β-strand separated from the rest of the domain by an intervening sequence termed the HOOK-domain, whose sequence varies between Ca_Vβ subunits. Here we have been guided by the recent structural studies of Ca_Vβ subunits in the design of specific truncated constructs, with the goal of investigating the role of the HOOK-domain of Ca_Vβ subunits in the modulation of inactivation of N-type calcium channels. We have co-expressed the β subunit constructs with Ca_V2.2 and α2δ-2, using the N-terminally palmitoylated β2a subunit, because it supports very little voltage-dependent inactivation, and making comparisons with β1b domains. Deletion of the variable region of the β2a HOOK-domain resulted in currents with a rapidly inactivating component, and additional mutation of the β2a palmitoylation motif further enhanced inactivation. The isolated GK-domain of β2a alone enhanced current amplitude, but the currents were rapidly and completely inactivating. When the β2a-GK-domain construct was extended proximally, by including the HOOK-domain and the ε-strand of the SH3-domain, inactivation was about 4 fold slower than in the absence of the HOOK domain. When the SH3-domain of β2a truncated prior to the HOOK-domain was co-expressed with the (HOOK+εSH3+GK)-domain of β2a, all the properties of β2a were restored, in terms of loss of inactivation. Furthermore, removal of the HOOK sequence from the (HOOK+εSH3+GK)-β2a construct increased inactivation. Together, these results provide evidence that the HOOK domain is an important determinant of inactivation.     

Electrostatic Influence on Ion Transport through the αHL Channel

The current-voltage relationship of a single Staphylococcus aureus alpha-hemolysin (alphaHL) channel is nonlinear, rectifying, and depends on the bulk pH and the ionic strength. The data are described qualitatively by a simple one-dimensional Nernst-Planck analysis in which the fixed charges inside and near the pore's entrances affect the transport of ions through the channel. The distances of these fixed charges from one of the channel's entrances are obtained from the channel's crystal structure. The model demonstrates that rectification of monovalent ion flow through the alphaHL channel can be related to the asymmetry in the location of the ionizable amino acid side chains.